1
SI Appendix April 12, 2014
A mechanistic rationale for targeting the unfolded protein response in pre-B
acute lymphoblastic leukemia
Behzad Kharabi Masouleh1,3, Huimin Geng1, Christian Hurtz1, Lai N. Chan1 Aaron C Logan2,
Mi Sook Chang4, Chuanxin Huang5, , Srividya Swaminathan1, Haibo Sun6, Valeria Cazzaniga1,7,
Giovanni Cazzaniga7, Elisabeth Paietta8, Ari Melnick5, H. Phillip Koeffler6, Markus Müschen1,2
Supplemental Material and Methods
Figures S1-S10
Tables S1-S5
2
SUPPLEMENTAL MATERIAL AND METHODS
Primary human samples and human cell lines
We obtained primary cases (Table S1) in compliance with the IRB of the University of California
San Francisco. The human Ph+ ALL, lymphoma or multiple myeloma cell lines were obtained
from DSMZ, Braunschweig, Germany. We maintained human leukemia cells in Roswell Park
Memorial Institute medium (RPMI-1640, Invitrogen, Carlsbad, CA) with GlutaMAX containing
20% fetal bovine serum, 100 IU ml−1 penicillin and 100 μg ml−1 streptomycin at 37°C in a
humidified incubator with 5% CO2. We cultured all primary human ALL cells on OP9 stromal
cells.
Extraction of bone marrow cells from mice
Bone marrow cells were extracted from young age-matched mice with different genetic
backgrounds (Table S2). We obtained the bone marrow cells by flushing cavities of femur and
tibia with PBS. After filtration through a 70 μm filter and depletion of erythrocytes using a lysis
buffer (BD PharmLyse, BD Biosciences), washed cells were either frozen for storage or subjected
to further experiments.
Mouse model of human Ph+ ALL
We collected bone marrow cells from the above mentioned mice and retrovirally transformed them
using BCR–ABL1 or NRASG12V in the presence of 10 ng ml−1 interleukin-7 (Peprotech) in
RetroNectin- (Takara) coated Petri dishes as described below. We maintained all BCR–ABL1-
transformed ALL cells derived from bone marrow of mice in Iscove’s modified Dulbecco’s
medium (IMDM, Invitrogen) with GlutaMAX containing 20% fetal bovine serum, 100 IU
ml−1 penicillin, 100 μg ml−1 streptomycin and 50 µM 2-mercaptoethanol. BCR–ABL1 or NRAS-
G12V-transformed ALL cells were propagated only for short periods of time and usually not longer
than for 2 months to avoid acquisition of additional genetic lesions during long-term cell culture.
BCR-ABL1 transformed pre-B cells were transduced with different empty vector controls (EV)
and either (4-OHT)-inducible or non-inducible Cre and deleted by Cre-mediated activation. A
detailed overview of the used plasmids is shown in Table S5, For in vivo experiments ALL cells
were then labeled with lentiviral firefly luciferase (D.B. Kohn, University of California Los
Angeles, Los Angeles, CA) and injected via tail into sublethally irradiated (250 cGy) NOD/SCID
recipient mice. Engraftment was monitored using luciferase bioimaging (VIS 100
bioluminescence/optical imaging system; Xenogen). D-Luciferin (Xenogen) dissolved in PBS was
injected intraperitoneally at a dose of 2.5 mg per mouse 15 min before measuring the light
3
emission. Seven mice per group were injected via tail vein injection. When a mouse became
terminally sick, it was sacrificed and bone marrow and spleen were collected for flow cytometry
analysis. All mouse experiments were subject to approval by the Children’s Hospital Los Angeles
Institutional Animal Care and Use Committee.
Retroviral transduction
We performed transfections of retroviral constructs and their corresponding empty vector controls
(Table S5) using Lipofectamine 2000 (Invitrogen) with Opti-MEM media (Invitrogen). We
produced retroviral supernatants to infect murine cells by cotransfecting 293FT cells with the
plasmids pHIT60 (gag-pol) and pHIT123 (ecotropic env; provided by D.B. Kohn, University of
California, Los Angeles, Los Angeles, CA). To infect human cells we replaced pHIT123 with
pHIT456 (amphotropic env; provided by D.B. Kohn, University of California, Los Angeles, Los
Angeles, CA). Cultivation was performed in high glucose DMEM (Invitrogen) with GlutaMAX
containing 10% fetal bovine serum, 100 IU ml−1 penicillin, 100 µg ml−1 streptomycin, 25 mM
Hepes, 1 mM sodium pyruvate, and 0.1 mM of nonessential amino acids. We replaced regular
media after 16 h with growth media containing 10 mM sodium butyrate. After 8 h of incubation,
the media was changed back to regular growth media. 24 h later, the virus supernatants were
harvested, filtered through a 0.45 µm filter, and loaded by centrifugation (2,000 g for 90 min at
32°C) two times on 50 µg ml−1 RetroNectin-coated non-tissue 6-well plates. 2–3 × 106 pre–B cells
were transduced per well by centrifugation at 500 g for 30 min and maintained overnight at 37°C
with 5% CO2 before transferring into culture flasks.
Senescence-associated β-galactosidase assay
Senescence-associated β-galactosidase activity was performed on cytospin preparations as
described (1). Briefly, a fixative solution (0.25% glutaraldehyde, 2% paraformaldehyde in PBS
pH 5.5 for mouse cells) was freshly generated. To this end, 1 g paraformaldehyde was dissolved
in 50 ml PBS at pH 5.5 by heating followed by addition of 250 μl of a 50% stock glutaraldehyde
solution. 1× X-gal staining solution (10 ml) was prepared as follows: 9.3 ml PBS/MgCl2, 0.5 ml
20× KC solution (that is, 820 mg K3Fe(CN)6 and 1,050 mg K4Fe(CN)6 × 3H2O in 25 ml PBS) and
0.25 ml 40× X-gal (that is, 40 mg 5-bromo-4-chloro-3-indolyl β-D-galactoside per milliliter
of N,N-dimethylformamide) solutions were mixed. For BCR–ABL1-transformed ALL cells,
100,000 cells per cytospin were used (700 r.p.m., 8 min). The fixative solution was pipetted onto
cytospins and incubated for 10 min at room temperature, then washed twice for 5 min in
4
PBS/MgCl2. Cytospin preparations were submerged in 1× X-gal solution, incubated overnight at
37 °C in a humidified chamber and washed twice in PBS.
Single-locus quantitative ChIP, ChIP-on-chip and ChIP-seq analysis
Cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature, lysed, and
DNA was sonicated to a fragment size of 150-350bp. After sonication, immunoprecipitations were
performed with the following antibodies: anti-BCL6 (N3, Santa Cruz), anti-BACH2, or rabbit
control IgG (ChIP-grade, Abcam). DNA fragments enriched by ChIP were identified by
quantitatively PCR (ChIP-QPCR) using the Fast SYBR green kit. The GAPDH gene, which is not
a BCL6/BACH2 target, was used as negative control. The enrichment for each antibody at
examined loci is expressed as percentage of input. Primers are listed in Table S3. ChIP-seq
libraries were prepared using Illumina ChIP-seq Library preparation Kit according to the
manufacturer’s instructions with 10ng of purified ChIP DNA and sequenced for 36 cycles using
Illumina GAIIx sequencer. Peak calling and downstream analysis was performed using the
ChIPseeqer package (2). The ChIPseq data have been uploaded to GEO under accession number
GSE44420. ChIP-on-Chip was performed in three Ph+ ALL cell lines in duplicates as described in
(1) (GSE24404). Briefly, to identify target genes of BCL6 from each array, the log-ratio between
the probe intensities of the ChIP product and input was computed by taking moving windows of
three neighboring probes for the average log-ratio and determining the maximum value of those
windows as the signal for each probeset. Random permutation probe log-ratios were used as
background control. The peak cutoff was established as higher than the 99th percentile of the
24,175 log-ratio values generated from random permutation probes on the array. A locus with
maximum moving average above the cut-offs in both replicates, or in one replicate but the Pearson
correlation coefficient of the probes signal of the promoter between the two replicates higher than
0.8 (a rescue procedure), is considered a binding target.
Gene expression - microarray and RNAseq analysis
Microarray. Total RNA from cells used for microarray was isolated by RNeasy purification.
Biotinylated cRNA was generated and fragmented according to the Affymetrix protocol and
hybridized to mouse 430A 2.0 Array (Affymetrix). After scanning (GeneChip Scanner 3000 7G;
Affymetrix), the generated .cel files were imported to Expression Console software (Affymetrix)
and processed using the RMA average algorithm for normalization and summarization. RNAseq.
Total RNA were extracted and subjected to high-throughput sequencing. Illumina multiplexing
library construction, HiSeq 2000 SR50 sequencing, and data generation were performed by
5
Epigenomics Core Facility at Weill Cornell Medical College (New York, NY) according to
manufacturer's instructions. Raw image data were converted into base calls and fastq files via the
Illumina pipeline CASAVA version 1.8 with default parameters. All 50-bp-long reads were
mapped to the reference mouse genome sequence, mm9, using Tophat with the default parameters
(Trapnell et al., 2009). The mRNA expression level for each gene was represented as log2 RPKM
(reads per kilobase per million reads), called by Seqgene (Deng X, BMC Bioinformatics 2011).
Patient outcome and gene expression microarray data
Gene expression microarray and patient outcome data were obtained from the GEO database
accession numbers GSE5314 (3) and GSE34941 (Geng et al., 2012) of the Eastern Cooperative
Oncology Group (ECOG) Clinical Trial E2993 for adult B-ALL, from GSE11877 and GSE28460
(4) of the Children’s Oncology Group (COG) Clinical Trial P9906 for children B-ALL (the
National Cancer Institute TARGET Data Matrix, http://target
nci.nih.gov/dataMatrix/TARGET_DataMatrix.html), from the St. Jude Research Hospital
pediatric ALL clinical trial (raw data can be downloaded from
http://www.stjuderesearch.org/site/data/ALL3/). The microarray raw data were normalized using
the RMA method with Expression ConsoleTM software HG-U133 A and B (St Jude data, n=132)
or NimbleScan software (version 2.5, Roche NimbleGen, Madison, WI) for the NimbleGen arrays
HG18 60mer expression 385K platform (ECOG data). Expression level of a gene in a sample was
determined by the average of expression values from multiple probe sets on the array representing
this gene. The P-values of differential expression of a gene between BCR-ABL1 ALL and normal
pre-B samples and other ALL subtypes were determined by two-sided Wilcoxon test. The end
points of the clinical data include overall survival (OS), relapse-free survival (RFS), risk, time to
relapse, and expression levels in samples at the time of relapse vs. expression levels at diagnosis.
Array-based methylation analysis using HELP
The HELP (HpaII tiny fragment enrichment by ligation mediated PCR) assay was performed as
previously published (Geng et al., 2012). One microgram of high molecular weight DNA was
digested overnight with isoschizomer enzymes HpaII and MspI (NEB, Ipswich, MA). DNA
fragments were purified using phenol/chloroform, resuspended in 10mM Tris-HCl pH 8.0, and
used immediately to set up the ligation reaction with MspI/HpaII-compatible adapters and T4 DNA
ligase. Ligation-mediated PCR was performed with enrichment for the 200 to 2000 base pair (bp)
products and then submitted for labeling and hybridization onto a human HG_17 promoter custom-
designed oligonucleotide array covering 25,626 HpaII amplifiable fragments within the promoters
6
of the genes. HELP data analysis was performed as described previously (5), using R software and
Bioconductor package (http://www.bioconductor.org/). Basically, raw data (.pair) files were
generated using NimbleScan software. Signal intensities at each HpaII amplifiable fragment were
calculated as a robust (25% trimmed) mean of their component probe-level signal intensities. Any
fragments found within the level of background MspI signal intensity, measured as 2.5 mean-
absolute- deviation (MAD) above the median of random probe signals, were considered as “failed”
probes and removed. A median normalization was performed on each array by subtracting the
median log-ratio (HpaII/MspI) of that array (resulting in median log-ratio of 0 for each array).
References:
1. Duy C et al. (2011) BCL6 enables Ph+ acute lymphoblastic leukaemia cells to survive BCR-
ABL1 kinase inhibition. Nature 473:384–388.
2. Giannopoulou EG, Elemento O (2011) An integrated ChIP-seq analysis platform with
customizable workflows. BMC Bioinformatics 12:277.
3. Juric D et al. (2007) Differential gene expression patterns and interaction networks in BCR-
ABL-positive and -negative adult acute lymphoblastic leukemias. J Clin Oncol Off J Am Soc
Clin Oncol 25:1341–1349.
4. Hogan LE et al. (2011) Integrated genomic analysis of relapsed childhood acute
lymphoblastic leukemia reveals therapeutic strategies. Blood 118:5218–5226.
5. Geng H et al. (2012) Integrative epigenomic analysis identifies biomarkers and therapeutic
targets in adult B-acute lymphoblastic leukemia. Cancer Discov 2:1004–1023.
Fig. S1: Validation of Hspa5 (Ig heavy chain binding protein) deletion
Cre
b-actin
Hspa5
EV Cre Hspa5fl/fl ALLEV Cre
2 days 3 days
aHspa5fl/fl pre-B ALL +EVGFP
94 32 61 86
EV-GFP
CD
45.2
I II III IV
99 96 97 97
I II III IV
Cre-GFP
CD
45.2
Hspa5fl/fl pre-B ALL +CreGFP
b
Hspa5fl/fl pre-B ALL+EV +Cre Hspa5#1 2 3 4 5 6 7 8 9 #1 2 3 4 5 6 7 8 9 genotype
WTfloxed
deleted
c
Figure S1 legend: The protein levels of Hspa5 and Cre are measured by Western Blot analyses inHspa5fl/fl pre-B ALL cells with EV and Cre after two and three days of 4-OHT treatment using β-actinas loading control (a). Bone marrows and spleens from transplant recipient mice carrying Hspa5fl/fl
pre-B ALL with either CreGFP or EVGFP were stained for the surface markers CD45.1- and CD45.2+. Arepresentative analysis is shown (n=4) (b). A genomic PCR amplifying either the floxed allele(floxed), wildtype (WT) or after deletion (deleted) using a wildtype (C57BL6J), H2O and in vitrodeletion controls is shown for NOD/SCID mice (n=9) transplanted with Hspa5fl/fl pre-B ALL witheither CreGFP or EVGFP (c).
Cox6a2
Ccc
r7 m
RN
A le
vels
[%
Hprt
]
EV CreHspa5fl/fl EV Cre
60
50
40
30
20
10
0
Ccr7
1.6
Co
x6a2 m
RN
A [
% H
prt
]
EV CreHspa5fl/fl EV Cre
1.4
1
0.8
0.6
0.4
0.20
1.2
1.8
Ccn
g2 m
RN
A le
vels
[%
Hprt
]
EV CreHspa5fl/fl EV Cre
60
50
40
30
20
10
0
P=0.0007 P=0.001
Ccng2
Ccy
5rl m
RN
A le
vels
[%
Hprt
]
EV CreHspa5fl/fl EV Cre
10
8
6
4
2
0
Cy5rl
CD
22 m
RN
A le
vels
[%
Hprt
]
EV CreHspa5fl/fl EV Cre
20
15
10
5
0
P=0.0006 P=0.002
CD22
70P=0.001 P=0.01P=0.0003 P=0.003
P=0.0001 P=5.64 x 10-5
Fig, S2: Validation of microarray gene expression analysis of Hspa5-deletion
Hspa5fl/fl BCR-ABL1 ALL cells
b
a
c
e
d
Fig. S2 legend: The mRNA levels of Cox6a2, Ccng2, CD22, Ccr7 and Cy5rl were measuredby qRT-PCR in Hspa5fl/fl pre-B ALL cells transduced with empty vector control (EV) and 4-OHT-inducible retroviral Cre (Cre) after two days of 4-OHT treatment using Hprt as areference gene (n=2; a-e)
0
100
200
300
0
200
400
600
800
0
2000
4000
6000
8000
10000
12000
14000
XBP1
ERN1
HSPA5
0
500
1000
1500
2000PRDM1
mR
NA
leve
ls [
sig
na
l in
tensi
ty,
Aff
yme
trix
Ge
ne
Ch
IPa
rra
ys]
Xbp
1s
mR
NA
leve
ls [
% H
prt
]
EV µHC Ighm-/-0.00
0.05
0.10
0.15
0.20
P=0.0002Xbp1s
Xbp
1s m
RN
A leve
ls [
% H
prt
]
0
10
20
30
40
50
60
Fig. S3: Expression of plasma cell-specific unfolded protein response molecules ERN1, HSPA5, PRDM1 and XBP1 in normal pre-B cells and pre-B ALL
a
b
c
d
e
f
g
PRDM1 P=2.61 x 10-7
Log H
paII
/MspI
4
2
0
-2
PRDM1 P=0.0004
Gene
expre
ssio
n v
alu
es
for
PR
DM
1
4
4.5
5
XBP1 P=1.63 x 10-7
Log H
paII
/MspI
4
2
0
-2
3
1
-1
Gene
expre
ssio
n v
alu
es
for
XB
P1
XBP1 P=0.0014
6
8
7
9
10
ERN1 P=0.0001
Log H
paII
/MspI
1
0
-1
-2
Gene
expre
ssio
n v
alu
es f
or
ER
N1
4.6
5
5.4
5.8
ERN1 P=7.7 x 10-7
HSPA5 P=1.8 x 10-4
Log H
paII
/MspI
4
2
0
-2
Gene
expre
ssio
n v
alu
es
for
HS
PA
5
5.5
6.0
6.5
7.0
HSPA5 P=0.018
5.0
Pre-B cells/Pre-B ALL B cell lymphoma Pre-B cells/Pre-B ALL Mature B cells/B cell lymphoma
h
i
j
k
Xbp1s
Fig. S3 legend: The gene expression profiling of UPR-related genes (ERN1 (IRE1), HSPA5 (BiP),PRDM1 (Blimp1) and XBP1 microarray data (GEO accession numbers GSE45460, GSE19599,GSM488979, GSE13411, GSE12453 is shown (a). Xbp1s mRNA levels were measured by qRT-PCRusing specific primers in Pro-B and Pre-BI cells using sorted Lin- Sca-1+ c-Kit+ (LSK) multilineageprogenitors, and bone marrow B cell precursor populations as well as in mature B cells from spleens ofC57BL/6 mice (n=3; b). Xbp1s mRNA levels were measured by qRT-PCR after reconstitution of BCR-ABL1 Ighm-/- pre-B ALL cells with an Ig µ-heavy chain expression vector (µHC) and empty vectorcontrol (EV; n=3; c). The methylation of the promoter regions of ERN1, HSPA5, PRDM1 and XBP1 genesin ALL subsets compared to mature B cell Non-Hodgkin lymphomas and pre-B cells was assessed byHELP assay (GEO number GSE34937) and the p-values were calculated from Wilcoxon test (d-g). themRNA levels of ERN1, HSPA5, PRDM1 and XBP1 genes from gene expression microarray data indifferent ALL subsets in comparison to B-cell subsets, Hodgkin and B-NHL and the p-value wascalculated by Wilcoxon test (St Jude http://www.stjuderesearch.org/data/ALL3; h-k)
Fig. S4: Sorting strategies of different human and murine B-cell fractions
CD43
B220
CD24B
P-1
NK1.1 + GR1
FS
C
IgM
IgD
Lineage
FS
C
Sca-1
c-k
it
Fig. S4 legend: Multilineage progenitors, and bone marrow Bcell precursor populations as well as mature B cells from spleensof C57BL/6 mice and healthy human donors were sortedaccording to Hardy fractions A-F using multiple surface markersincluding B220, CD43, BP-1, CD24, NK1.1/Gr1, IgD, IgM,Lineage, c-kit and Sca-1 with a detailed gating strategy as shownin the figure (n=5.a). The different fractions of MACS-enrichedCD19+ pre-B cells (original mononuclear cells: MNC; flow-through and positively selected CD19+ cells: CD19+ MACS)were stained with the surface markers CD22 and CD19 withthree patient-derived pre-B ALL cases (ALL-X2; ALL-Q5; ALL-O2) as a positive control (n=3; b).
CD19
CD
22
MNC Patient-derived pre-B ALL
Flow-through
CD19+ MACS
a
b
0.35
Prd
m1
mR
NA
leve
ls [%
Hprt
]
0.3
0.25
0.2
0.15
0.1
0.05
0
P=0.007 P=0.001
2.5
Xb
p1s
mR
NA
leve
ls [%
HP
RT
]
2
1.5
1
0.5
0
P=0.0005 P=0.002
0.35
0.3
0.25
0.2
0.15
0
0.1
0.05
Prdm1fl/fl ALL
Cre EV Cre
2 days 5 days
EV Cre EV Cre
2 days 5 days
EV
Prdm1fl/fl ALL
Multiple Myeloma
CD19
CD
138
Patient-derivedPre-B ALL
7 AAD
An
nexi
nV
18
19
EV
Cre
Day 1 Day 2 Day 5
14
20
18
25
Prdm1fl/fl ALL
51 XBP1s
b-actin
BM CD19+ pre-B cells Patient –derived Pre-B ALL
PRDM1
Fig. S5: Prdm1 (Blimp1) is required for proliferation and survival of pre-B ALL cells
a
c d e
f
7 AAD
Brd
US 39
S 24
G2/M 3.5
G0/1 57
G0/1 71.5
G2/M 4.3
100
80
60
40
20
0
P=0.0001
S phaseDay 5
Prdm1fl/fl ALL
EV
Cre
EV Cre
G2/MS phase
G0/1
g
200300
WT WT EV Cre EV Cre H20bp
Prdm1fl/fl ALLb
Prdm1flox
Prdm1
24 48 hours
Cre EV Cre
Day 1 Day 2
EV Cre
Day 5
Nu
mber
of
ap
opto
tic c
ells
[%
]
P=0.25 P=0.0004 P=0.0002
0
5
10
15
20
25
EV
Figure S5 legend: The protein levels of PRDM1 and spliced XBP1 (XBP1s) were measured by Westernblot analysis in MACS-enriched normal human CD19+ pre-B cells compared to patient-derived pre-BALL cells and analyzed by densitometry (ALL-Q5, ALL-LAX9, ALL-X2, ALL-BLQ11, ALL-O2) usingβ-actin as loading control (n=3; a). Genomic deletion was assessed after 24 hours treatment with 4-OHTusing specific primers to amplify the floxed allele in Prdm1fl/fl ALL cells transduced with empty vectorcontrol (EV) and 4-OHT-inducible retroviral Cre (Cre) using a wildtype (WT) and H2O control (n=2)(b). Prdm1 mRNA levels were assessed in Prdm1fl/fl ALL cells with EV and Cre at 2 and 5 days aftertreatment with 4-OHT (n=3) (c). Likewise, the Xbp1s mRNA levels were measured in Prdm1fl/f ALLcells with EV and Cre by qRT-PCR after 2 and 5 days post 4-OHT treatment (n=3; d). A representativeFACS blot is shown for a patient-derived Pre-B ALL sample (ALL-ICN1) and a multiple myeloma cellline (JJN3) stained for the surface markers CD138 and CD19 (e). Apoptosis was assessed by Annexin-Vand 7AAD FACS staining in Prdm1fl/f ALL cells with EV and Cre after 1, 2 and 5 days treatment with 4-OHT (n=3; f). Prdm1fl/f ALL cells with EV and Cre were stained with BrdU and 7AAD and thepercentages of cells in the G0/1, S and G2/M cell cycles are indicated after 5 days of 4-OHT treatment andshown in bar graph (n=3, p=0.0001 for the S-Phase; g).
XB
P1s
Ra
tio D
ensi
tom
etr
y [%
]
P=0.03
0
1
2
3
4
PR
DM
1 R
atio
Densi
tom
etr
y [%
]
P=0.06
0
1
2
3
4
Fig. S6: Regulation of XBP1 in Ph+ ALL
Input
BACH2
0
BCL6
16
0
16
0
XBP1
16
0
2 kb
BACH2, BCL6 ChIP-seq; XBP1 promoter
Log r
atio
BC
L6 C
hIP
/ In
put
XBP1
Imatinib
0 mmol/l
HPRT HPRT
Human Ph+ ALL, BCL6 ChIP-on-chip
-1 -0.5 -0 kb -1 -0.5 -0 kb -1 -0.5 -0 kb from TSS
-1 -0.5 -0 kb -1 -0.5 -0 kb -1 -0.5 -0 kb from TSS
0 mmol/l
2
1
0
2
1
0
-1 -0.5 -0 kb -1 -0.5 -0 kb -1 -0.5 -0 kb from TSS
-1 -0.5 -0 kb -1 -0.5 -0 kb -1 -0.5 -0 kb from TSS
Imatinib
10 mmol/l
10 mmol/l
XBP1
2
1
0
2
1
0
0 10 0 10 0 10 0 10 IM [µM]
b-actin
BACH2
Human Ph+ ALL Mouse BCR-ABL1pre-B ALL
c
d
Fig S6 legends: Protein levels of phosphorylated and total ERK were measured by Western blotanalysis in human Ph+ ALL cells (PDX2, LAX7R) treated with or without the MEK-inhibitorPD325901 for 5 hours (50 nM PD325901) using β-actin as loading control (a). Protein levels ofphosphorylated and total S6 were measured by Western blot analysis in human Ph+ ALL cells (PDX2,LAX7R) treated with or without the AKT-inhibitor AZD5363 for 5 hours (10 μM AZD5363) using β-actin as loading control (b). Protein levels of BACH2 were measured by Western blot analysis inhuman Ph+ ALL cells (ICN1, BV-173, TOM1) and mouse BCR-ABL1 pre-B ALL cells treated with orwithout the TKI imatinib for 16 hours (human: 10 µM IM; mouse 2 µM IM) using β-actin as loadingcontrol (c). ChIP-seq analysis was performed using antibodies against BACH2 and BCL6 (GEOnumber GSE44420). Peaks with significant enrichment of ChIP-seq relative to input were identified byChIP-seeqer (http://physiology.med.cornell.edu/faculty/elemento/lab/chipseq.shtml) and shown as boldunderlines. The gene model in UCSC genome browser view (hg18) and the arrow for transcription startsite were shown (d). BCL6 ChIP-on-chip analysis was performed on human Ph+ALL cell lines (Tom1,Nalm1, BV-173) treated with and without imatinib (10 µmol-l) for 24 h as previously described (26)(two representatives shown; GEO number GSE24404) (e).
Human Ph+ ALL
0 50 0 50 PD325901 [nM]
β-actin
ERKpT202/Y204
ERK
a
Human Ph+ ALL
0 10 0 10 AZD5363 [μM]
β-actin
S6 pS235/36
S6
b
e
Fig. S7: Verification of Xbp1-deletion in pre-B cells and BCR-ABL1 ALL
+/+ +/+ EV Cre EV Cre E V Cre EV Cre EV Cre EV Cre H20(bp)
400500
Xbp1fl/fl BCR-ABL1 ALL cells
a
EV Cre0
0.4
0.8
1.2
1.6
2
P=0.0049
Xbp1 m
RN
A le
vels
[%
HP
RT
]
Xbp1 m
RN
A le
vels
[%
HP
RT
]
0
1
2
3
4
5
6
P=0.007
EV Cre
b cXbp1fl/fl pre-B cells Xbp1fl/fl BCR-ABL1 ALL cells
Fig. S7 legend: A representative schematic of the designed primers amplifying the loxP sites at exon 2
of the Xbp1 gene is shown in the top panel. In the lower panel, genomic deletion by PCR was assessed
in Xbp1fl/fl ALL cells with EV or Cre after 1 day administration of 4-OHT using a wildtype (WT) and
H2O control (n=6; a). Xbp1 mRNA levels were measured in IL7-dependent Xbp1fl/fl pre-B cells with
EV or Cre after 24 hours of 4-OHT treatment by qRT-PCR (n=3; b). Xbp1 mRNA levels were
measured in Xbp1fl/fl BCR-ABL1 ALL cells with EV and Cre after 24 hours of 4-OHT treatment by
qRT-PCR (n=3; c). Xbp1fl/fl BCR-ABL1 ALL cells were stained for the surface markers CD19, B220, c-
Kit, Sca-1 and CD13 (n=3; d). Apoptosis was quantified by Annexin-V and 7AAD staining in Xbp1fl/fl
NRASG12D ALL cells with EV and Cre after 1, 4 and 7 days treatment with 4-OHT (n=3; e) Apoptosis
was quantified by Annexin-V and 7AAD FACS staining in IL7-dependent Xbp1fl/fl pre-B cells with
(Cre) or without (EV) deletion of Xbp1 after 1, 2 and 5 days treatment with 4-OHT (n=3; f).
B220
CD
19
Sca1
c-K
it
CD13
Sca
-1
Xbp1fl/fl BCR-ABL1 ALL cellsd
Day 1 Day 4
EV Cre
Day 7
Nu
mbe
r of apo
pto
tic c
ells
[%
]
0
5
10
15
20
25
EV CreEV Cre
P= 0.0002 0.001 3.5 X 10-5
30
eXbp1fl/fl NRASG12D ALL
Day 1 Day 4
EV Cre
Day 7
Nu
mbe
r of apo
pto
tic c
ells
[%
]
0
20
40
60
EV CreEV Cre
P= 0.0008 0.06 1.57 X 10-5
80
Xbp1fl/fl Pre-B cells
f
Fig. S8: Pharmacological inhibition of ERN1 endoribonuclease activity causes cell death in patient-derived pre-B ALL cells
Dose
Eff
ect
0 20 40 60 STF-083010 concentrations [μM]
0
20
40
60
80
100Day 0 Day 4
Control
TN 10 µg
ERN1i 5 µM
ERN1i 10 µM
ERN1i 15 µM
ERN1i 30 µM
ERN1i 60 µM
FSC
DA
PI
da Patient-derived Pre-B ALL cells Patient-derived Pre-B ALL cells
Fig. S8 legend: The dose effect was calculated as measuredfor mRNA levels of XBP1s by qRT-PCR with the CalcuSynsoftware in patient-derived pre-B ALL cells (ALL-Q5) after24 hours treatment with either the ER-stress inducerTunicamycin (TN; 10 µg) in combination with STF-083010(0-60 µM) (ERN1i) for 24 hours (n=3; a). Patient-derived pre-B ALL cases (ALL-Q5 and ICN1) were treated with STF-083010 or A106 (0-60 µM) and the relative viability wasassessed by CCK-8 assay (n=3; b). Apoptosis was assessed byAnnexin-V and 7AAD FACS staining in two patient-derivedpre-B ALL cases (ALL-Q5 and ALL-O2) after 3 daystreatment with either STF-083010 and A106 at twoconcentrations (30 and 60 µM) or DMSO control (0 µM; n=3;c). Patient-derived pre-B ALL (ALL-Q5) were treated withA106 (0-60µM) and TN (10µg) and viability was assessed byDAPI staining up to four days. The representative FACS blotsare shown at day 0 and 4 (n=3; d).
0 10 20 30 40 50 60 ERN1 inhibitor [mM]
80
60
40
20
0
Su
rviv
al o
f ce
lls [%
] Pre-B ALL 1 + STF-083010Pre-B ALL 1+ A106Pre-B ALL 2 + STF-083010Pre-B ALL 2 + A106
b
7 AAD
Annexi
nV
0STF-08301030 mM
A10630 mM
6 69 67
18 71 61
Pre
-B A
LL
Pre
-BA
LLc
Nu
mb
er
of apo
pto
tic c
ells
[%
]
0
20
40
60
80
Ctr STF-083010
A106 Ctr STF-083010
A106
3.5 X 10-5 5.3 X 10-5
Fig. S9: Pharmacological inhibition of ERN1 endoribonuclease activity causes cell death in patient-derived pre-B ALL cells
17 66 94
B-NHL,Mantle cell lymphoma
22 38 66
7 31 87
12 86 94
15 34 65
11 76 96
8 7 9
7 AAD
Annexi
nV
6 25 81
ERN1i [μM] 0 30 60
B-NHL,DLBCL
Multiple Myeloma
Pre-B ALL
Pre-B ALL
Pre-B ALL
Ph+ ALL
Ph+ ALL
a
Fig. S9 legend: Apoptosis was assessed by Annexin-V and 7AAD FACS staining in a panel of humancell lines including B-NHL (Toledo, Jeko), multiple myeloma (JJN3) and patient-derived Pre-B and Ph+
ALL cases (BLQ1, ICN1, ALL-O2, ALL-L3, ALL-Q5) after 3 days treatment with STF-083010 at twoconcentrations (30 and 60 µM) or DMSO control (n=3; a)). Patient-derived pre-B ALL cells (ALL-Q5)were treated with either TN (10µg) or STF-083010 (0-60 µM) and stained with ER-Tracker green after24 hours and 48 hours (n=3; b).
TN
DMSO
A106 5 µM
A106 10 µM
A106 15 µM
A106 30 µM
A106 60 µM
48 h24 h
Patient-derived Pre-B ALLb
Fig. S10: High XBP1 mRNA levels at the time of diagnosis predict poor clinical outcomein patients with pre-B ALL
COG P9906 n=207 pre-B ALL
XBP1Low
XBP1High
P=0.005
Time after diagnosis [years]
100
80
60
40
20
0
0 1 2 3 4 5 6
Rela
pse
-fre
e s
urv
iva
l [%
]
XBP1Low BACH2High
XBP1High BACH2Low
COG P9906 n=207 pre-B-ALL
P=0.00064
Time after diagnosis [years]
100
80
60
40
20
0
0 1 2 3 4 5 6
XBP1 probe set COG: Affy plus2, 200670_at: 11 probes
ECOG, XBP1, NimbleGen: HSAP0406S00034989
ER
N1 L
og
2e
xpre
ssio
n v
alu
es
CR+ CR-
n=22 n=14
n=53 adult pre-B ALL
ECOG E2993, ERN1 mRNA levels
P=0.04 P=0.01
n=36 adult Ph+ ALL
1.5
1
0.5
0
-0.5
-1
1.5
1
0.5
0
-0.5
-1
-1.5
CR+ CR-
n=38 n=15
ER
N1 L
og
2e
xpre
ssio
n v
alu
es
Rela
pse
-fre
e s
urv
iva
l [%
]
b
a
c d
e f Fig. S10 legend: The position ofthe different used XBP1 probesetin this study is shown at the3’UTR for the Affy plus2 (11probesets) and NimbleGen (1probeset; a, b). In an analysis,pre-B ALL (COG clinical trialP9906, n=207) patients weresegregated into two groups basedon high or low mRNA levels inrespect to the median mRNAvalue of the XBP1 probeset (c).
In a multivariate analysis, pre-B ALL (COG clinical trial P9906, n=207) patients were segregatedinto two groups based on high or low mRNA levels of XBP1 or BACH2, in respect to the medianexpression value of the XBP1 probeset. (d). ERN1 expression was measured in patients with either apositive (CR+) or negative complete response (CR-) in both adult Ph+ ALL (n=36) and adult pre-BALL patients (n=53) (e, f).
1
Table S1: Overview of patient-derived samples of Ph+ ALL and cell lines __________________________________________________________________________ Case Karyotype Clinical course Gender/Age ___________________________________________________________________________________________ Primary cases, Pre-B ALL ALL-X2 uncharacterized Unknown m/38
LAX9 t(9;22)(q34;q11) At diganosis m
del(12)(p12;p13)
ALL-O2 NRASG12D At diagnosis m/7
BLQ1 FISH der(9), der(22) Unknown
ALL-Q5 KRASG12V Unknown f
BLQ11 FISH der(9), der(22) Relapse (Imatinib) m
ALL-L3 KRASG12V At diagnosis
ICN1 der(9)(q10)t(9;22)(q34;q11) At diagnosis f
LAX7R uncharacterized Relapse
PDX2 BCR-ABL1 p190 At diagnosis f/52 Primary cases, Multiple myeloma MM1 n.d. Newly diagnosed m/44
MM2 n.d. Relapsed m/66
MM3 n.d. Persistent /Relapsed m/66
MM4 n.d. Persistent /Relapsed m/68
MM5 n.d. Newly diagnosed f/36
MM6 n.d. Persistent, at diagnosis m/65 ___________________________________________________________________________________________
Cell lines BV173 add(1)(q42), add(8)(p23), m/45
der(22)t(9;22)(q34;q11)
Nalm1 der(7)t(7;9;15)(q10;?;q15), f/3
t(9;9)(p24;q33-q34) t(9;22)
(q34;q11), der(15)t(7;9;15)
SUP-B15 t(1;1)(p11;q31), add(3)(q27), Relapse m/9
add(10)(q25), t(9;22)(q34;q11)
TOM1 del(7)(p14),der(9)del(9)(q13q34) Refractory f/54
der(22)t(9;22)(q34;q11)
JJN-3 hypotriploid karyotype At diagnosis f/57
with 9% polyploidy
JeKo-1 ATM amplification, f/78
MYC amplification
P16INK4A deletion
SYK amplification
P53 deletion/mutation
Toledo Complex karyotype f ___________________________________________________________________________________________ Notes: All primary samples are bone marrow biopsies, blast content >80%
2
Table S2: Overview of mouse strains used in this study
_________________________________________________________________________________ Mouse strain Source Purpose _________________________________________________________________________________ Bach2-/- Kazuhiko Igarashi, Tohoku University Analysis of Xbp1 as Bach2 target gene
BCL6 -/- B. Hilda Ye, Albert Einstein College Analysis of Xbp1 as BCL6 target gene aXbp1fl/fl Laurie H. Glimcher, Cornell Medical School Genetic loss-of-function experiments bStat5fl/fl Lothar Hennighausen, NIDDK Analysis of Xbp1 as STAT5 target gene Prdm1fl/fl Jackson Laboratories Genetic loss-of-function experiments Hspa5 fl/fl Amy S Lee, USC Genetic loss-of-function experiments
NOD/SCID Jackson Laboratories Xenograft recipient mice ___________________________________________________________________________________________ Notes: a Lee, A.-H., Scapa, E. F., Cohen, D. E. & Glimcher, L. H. Regulation of hepatic lipogenesis by the transcription
factor XBP1. Science 320, 1492–1496 (2008). b Liu, X. et al. Stat5a is mandatory for adult mammary gland development and lactogenesis. Genes Dev. 11, 179–
186 (1997).
3
Table S3: Sequences of oligonucleotide primers used Quantitative RT-PCR
Human
COX6B_F 5’-AACTACAAGACCGCCCCTTT-3’
COX6B_R 5’-GCAGCCAGTTCAGATCTTCC-3’
XBP1s_F 5’- TGCTGAGTCCGCAGCAGGTG -3’
XBP1s_R 5’- GCTGGCAGGCTCTGGGGAAG -3’
ERDJ4_F 5’- GCTACTCCCCAGTCAATTTTCA -3’
ERDJ4_R 5’- CCGATTTTGGCACACCTAAGAT -3’
p58IPK_F 5’- TGTGTTTGGGATGCAGAACTAC -3’
p58IPK_R 5’- TCTTCAACTTTGACGCAGCTT -3’
SEC61A1_F 5’- TGTCATCTCCCAAATGCTCTCA -3’
SEC61A1_R 5’- ACAGGTAATAGCAAAGGCCAC -3’
Mouse
Hprt_F 5’-GGGGGCTATAAGTTCTTTGC-3’
Hprt_R 5’-TCCAACACTTCGAGAGGTCC-3’
Xbp1 (ex2)_F 5’- CCTGAGCCCGGAGGAGAA -3’
Xbp1 (ex2)_R 5’- CTCGAGCAGTCTGCGCTG -3’
ERdj4_F 5’- GGATGGTTCTAGTAGACAAAGG-3’
ERdj4_R 5’- CTTCGTTGAGTGACAGTCCTGC-3’
p58IPK_F 5’- GCATCTTGAATTGGGGAAGA-3’
p58IPK_R 5’- CAAGCTTCCCTTGTTTGAGC-3’
Prdm1_F 5’- GAAATGGAAAGATCTATTCCAGAG-3’
Prdm1_R 5’- GGCATTCTTGGGAACTGTGT-3’
Cox6a2_F 5’- CGGTTATGAGCACCCTTGAT-3’
Cox6a2_R 5’- CTGTTCCCAAAGAGCCAGAG-3’
Ccng2_F 5’-TGATCCGCATCAGTCAGTGT-3’
Ccng2_R 5’- ACAATCGCGTGGTACAAGTG-3’
CD22_F 5’-GAGCAAGCTCACCTTCCAAC-3’
CD22_R 5’-CACCTCTGTGGGATTGACCT-3’
Ccr7_F 5’- GCCTTGATCACCATCCAAGT-3’
Ccr7_R 5’- GATCACCTTGATGGCCTTGT-3’
Cy5r1_F 5’- ATCCAACCCAGTGCTTTCTG-3’
Cy5rl_R 5’- CAAAGCCCTTGCTGTAGGTC-3’
Genomic PCR
Mouse
Xbpt_F 5’-TTTGGCTTGGGGAGGGACA-3’
Xbpt_R 5’-AGCAGTCTGCGCTGCTACTCT-3’
Prdm1_F 5’-CAATGCTTGTCTAGTGTC 3’
Prdm1_R 5’-AGTAGTTGAATGGGAGC-3’
Hspa5_F 5’-GATTTGAACTCAGGACCTTCGGAAGAGCAG-3’
Hspa5_R 5’-TTGTTAGGGGTCGTTCACCTAGA-3’
4
Table S3: Sequences of oligonucleotide primers used –continued
Single locus ChIP PCR
Human
BACH2_F CCTACCTGGCAAAAACAAAAAC
BACH2_R TCTTTTTGAGCAGTGGCATAGA
BCL6_F 5’-CCGAGAATTGAGCTCTGTTGA-3’
BCL6_R 5’- GGCAGCAACAGCAATAATCA-3
XBP1_F AATGTTTTGGGAGAAGTCTGGA
XBP1_R TAAAACGTAACAGGTGGCATGA
GAPDH_F ACGTAGCTCAGGCCTCAAGA
GADPH_R GCTGCGGGCTCAATTTATAG
5
Table S4: Antibodies used for Western blotting and flow cytometry
Western blot
Antigen Clone ID Source
ACTB Polyclonal (ab8227) Abcam
XBP1 Polyclonal (ab37152) Abcam
ARF- Mo Polyclonal (ab80) Abcam
P21 C-19 Santa Cruz Biotechnology
P27 F-8 Santa Cruz Biotechnology
p38 5F11 Cell Signaling Technology
Tp53 IC12 Cell Signaling Technology
p-P38(Thr180/Tyr182) Polyclonal (9211) Cell Signaling Technology
JNK Polyclonal (9252) Cell Signaling Technology
p-JNK(Thr183/Tyr185) Polyclonal (9251) Cell Signaling Technology
CHOP Polyclonal (5554) Cell Signaling Technology
Prdm1 Monoclonal (9115) Cell Signaling Technology
Cre Polyclonal (ab40011) Abcam
pERK(Thr202/Tyr) Monoclonal (2211) Cell Signaling Technology
ERK Polyclonal (ab40011) Abcam
pS6(Ser235/236) Polyclonal (2211) Cell Signaling Technology
S6 Monoclonal (2217) Cell Signaling Technology
Flow cytometry
Surface Antigen Source
CD19 BD Biosciences
Sca-1/Ly6a BD Biosciences
CD117/cKIT BD Biosciences
CD138/SDC1 BD Biosciences
B220/CD45R BD Biosciences
CD43 Biolegend
BP-1 Biolegend
NK1.1 and GR1 Biolegend
IgD Biolegend
IgM Biolegend
Lin Biolegend
CD24 BD Biosciences
Annexin-V BD Biosciences
7AAD Invitrogen
TruStain FxX block BD Biosciences
Lin (CD3/33/56) BD Biosciences
CD34 Biolegend
CD10 BD Biosciences
CD19 Invitrogen
CD22 BD Biosciences
Human Fc block Miltenyi
BrdU Invitrogen
6
Table S5: Retroviral and lentiviral vectors used
Constitutive expression Inducible activation MSCV BCR-ABL1-IRES-Neo MSCV-Cre-ERT2
MSCV-GFP-IRES MSCV-ERT2________________
MSCV-GFP-IRES-CRE MSCV-Cre-ERT2-GFP
MI µHC-CD8 MSCV-ERT2-GFP___________
MI CD8 PAX5-ERT2-GFP
pCCL-c-MNDU3-Luc-SV40-Neo pcl6-ERT2-GFP_____________